Nucleotide and protein sequence of mammastatin and methods of use

ABSTRACT

A nucleic acid sequence encoding Mammastatin, a specific mammary cell growth inhibitor. Mammastatin is encoded by a single nucleic acid sequence and has an approximate molecular weight of 44 kDa in its inactive, non-phosphorylated form. Normal mammary cells express functional phosphorylated forms having approximate molecular weights of 53 kDa and 49 kDa. Metastatic mammary cells either do not express Mammastatin at all, or do not express the 53 kDa or 49 kDa, phosphorylated forms. Mammary cancer cells are inhibited in their growth by the ?administration of phosphorylated Mammastatin.

BACKGROUND OF THE INVENTION

[0001] Breast cancer is a disease that kills over 45,000 women each yearin the United States alone. Over 180,000 new cases of breast cancer arediagnosed annually, and it is estimated that one in eight women willdevelop breast cancer. These numbers indicate that breast cancer is oneof the most dangerous diseases facing women today. Cancer research hasbeen unable to determine the cause of breast cancer, and has not found asuitable method of therapy or prevention.

[0002] A woman diagnosed with breast cancer may be treated with surgery,hormone therapy, chemotherapy, and radiation. If the patient developsmetastatic disease, radiation and high dose chemotherapy are required toablate the cancer in remote areas such as the brain, bone, and liver.

[0003] The current therapies available for the treatment of breastcancer are toxic, dangerous, costly, and many are ineffective,especially in the treatment of metastatic disease. The table below wasextracted from Churchill Livingston, Clinical Oncology, 1995, andsummarizes data available on the current methods of treatment andexpected survival rates. Treatment Method Effect Toxicity ResultSurvival adriamycin bolus kill cancer cells high can induce +14 monthsremission cyclophosphate bolus kill cancer cells high can induce +16months remission methotrexate infusion kill cancer cells high can induce+16 months remission 5F uracil infusion kill cancer cells high caninduce +18 months remission mix of above mixed kill cancer cells highcan induce +22 months remission taxol bolus kill cancer cells high caninduce +12 months remission estrogen oral may stop low can induce  +6months growth remission tamoxifen oral may stop low may stop +12 monthsgrowth progression mastectomy surgery remove tumor low may eliminate  +5years* cancer lumpectomy surgery remove tumor low may eliminate  +5years* cancer surgery and combination combination low may eliminate +7-10 years* tamoxifen cancer radiation mechanical kill cancer cellshigh can induce +14 months remission

[0004] Currently, there are no therapies that are effective for longterm treatment of breast cancer that has metastasized to lymph nodes ordistal sites. Local disease can be effectively treated by surgery, ifall of the cancer can be removed. A new therapy for the effectivetreatment of breast cancer that could stop the growth of breast cancerand of cells derived from metastatic cancer is urgently needed. Such atherapy would be useful in the treatment of localized breast cancer, inlong term treatment of metastatic disease, and as a follow-up treatmentafter surgical removal of tumors. Other applications include a growthinhibitor as a primary therapy and for preventative use.

[0005] Detection methods for breast cancer, such as mammogram, physicalexam, CAT-scan, and ultrasound, have significantly improved earlydetection of breast cancer. However, with these methods, a suspectedtumor must still be surgically removed for pathological examination todetermine if the tumor is benign or malignant, and to attempt todetermine the tissue type and grade of the malignancy. This pathologicaldiagnosis helps to determine what subsequent treatment protocols may beused.

[0006] For breast cancer, these methods are generally inconclusive, asadequate breast cancer tumor markers are not available. Availablemarkers such as CA 15-3 and CA 27-29 are used as indicators ofmetastases, however, they are not specific. There is a great need fordiagnostic tools and methods that can effectively and reliably diagnosebreast cancer, e.g., using new and specific breast cancer markers. Inaddition, a reliable and simple method for the early detection anddiagnosis of breast cancer is greatly needed. Preferably, such an earlydetection method would identify breast cancer in its early stages, trackprogression of breast cancer through advanced metastatic disease, anddiagnose the propensity of a patient to develop breast cancer or todevelop advanced disease. Most preferably, the diagnostic method couldbe used without tissue biopsy, e.g., by analysis of a body fluid such asblood.

[0007] Human mammary tissues undergo a burst of proliferative activitiesat the onset of menarche and during each menstrual cycle. Studies on theeffects of estrogen on mammary tissues and tumors indicate that estrogenis a primary growth-initiating factor for mammary tissues.Estradiol-sensitive growth factors have been characterized. In addition,mammary cell growth factors which are not hormonal in nature have alsobeen described.

[0008] Specific growth factors which have been shown to have astimulating effect on mammary tissue growth include platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-1) andtransforming growth factor (TGF) alpha. TGF-beta, on the other hand, hasbeen shown to suppress mammary tissue growth.

[0009] The regulation of mammary cell growth is of great importance inthe diagnosis and treatment of breast cancer. Neoplastic growth ofmammary tissues, if unchecked, can develop intouncontrollably-proliferating malignant tumors, which are the cause ofdeath of thousands of women yearly. A growth inhibition factor capableof specifically suppressing mammary cell growth would provide a dynamictool for use in the diagnosis and treatment of breast cancer.

[0010] Thus, it would be of great utility to isolate and characterize aspecific mammary cell growth inhibitor, to identify its nucleic acidsequence and amino acid sequence, and to recombinantly express theinhibitor as a purified protein. Diagnostic and therapeutic methodsusing the nucleic acid sequence and/or recombinantly produced inhibitorwould be of great utility in the diagnosis and treatment of breastcancer.

SUMMARY OF THE INVENTION

[0011] A specific mammary cell growth inhibitor, Mammastatin, has beenisolated from normal human mammary cells and characterized. It has nowbeen found that Mammastatin is produced by normal mammary cells, but notby breast cancer cells. Furthermore, it has now been found that thereduction or absence of Mammastatin in the blood correlate with thepresence of breast cancer. Administration of active Mammastatin preventsgrowth of breast cancer cells.

[0012] The nucleic acid sequence encoding Mammastatin has now beencloned, sequenced, and expressed recombinantly in host cells as anactive inhibitor of mammary cell growth. The isolated and characterizednucleic acid sequence (Sequence ID No: 1) and its deduced amino acidsequence (Sequence ID No: 2) provide unique and specific tools for usein the diagnosis and treatment of breast cancer.

[0013] The present invention provides an isolated and purified nucleicacid sequence encoding Mammastatin, a specific protein inhibitor ofmammary cell growth, and particularly of mammary cancer cell growth. Theinvention also includes plasmids and vectors containing the Mammastatinnucleic acid sequence, amino acid sequence of Mammastatin, and methods,kits, and compositions utilizing the Mammastatin nucleic acid or aminoacid sequences to produce purified mammary cell growth inhibitor and inthe diagnosis and treatment of breast cancer. The inventive compositionsinclude probes and primers that specifically hybridize to theMammastatin nucleic acid sequence and its RNA products.

[0014] The invention further includes a method for treating breastcancer by administering Mammastatin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a Western Blot showing expression of recombinantMammastatin in Eucaryotic Cos-7 cells.

[0016]FIG. 2 is an immunoblot showing expression of Mammastatin ininsect cells.

[0017]FIG. 3 is a graph showing inhibition of mammary cell growth byrecombinant Mammastatin produced by in vitro transcription andtranslation.

[0018]FIG. 4 is a graph showing growth inhibition in human mammarycancer cell growth by treatment with conditioned medium of Cos-7 cellstransfected with Mammastatin cDNA.

[0019]FIG. 5 is a Western Blot showing relative amounts of 53, 49 and 44kD Mammastatin in normal and cancerous human mammary cells.

[0020]FIG. 6 is an immunoblot showing phosphatase digestion ofMammastatin.

[0021]FIG. 7 is a graph showing the effect of phospatase on the activityof Mammastatin.

[0022]FIG. 8 is a Western Blot showing Mammastatin from normal andcancerous human mammary cells, as well as in mixed cultures of normaland cancerous cells.

[0023]FIG. 9 is a graph showing Mammastatin in normal human serum asanalyzed by ELISA.

[0024]FIG. 10 is a graph showing a Mammastatin ELISA standard curve.

[0025]FIG. 11 is a graph showing Mammastatin levels in breast cancerpatients over the course of treatment.

[0026]FIG. 12 is a Wesern blot showing expression of Mammastatin inducedby retrovirus.

[0027]FIGS. 13A, 13B and 13C are graphs showing the effect ofMammastatin treatment on MCF7 tumor cells in nude mice.

[0028]FIGS. 14A, 14B and 14C are graphs showing the effect ofMammastatin treatment on ______ tumor cells in nude mice.

[0029]FIG. 15 is a dot blot assay showing Mammastatin in blood fromnormal females versus the absence of Mammastatin in blood from breastcancer patients.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Mammastatin

[0030] Mammastatin is a protein growth inhibitor produced and secretedby normal human mammary epithelial cells. A mammary cell growthinhibitor was first described as an inhibitory protein activity presentin media conditioned by the growth of normal human mammary cells. Theinhibitory activity was identified in conditioned medium from normalhuman mammary cells, but not in media conditioned by the growth of humanmammary cancer cells. The inhibitory activity was determined by bioassayand antibody development to reside in three proteins, having theapproximate molecular weights of 53, 49 and 44 kDa (Ervin, Paul R.,Doctoral Dissertation University of Michigan, 1995).

[0031] It has now been determined that a specific mammary cell growthinhibitor, Mammastatin, is expressed as a 44 kD protein which isphosphorylately increasing the molecular weight to 49 kD and 53 kD. Thenon-ph 44 kD form is not an active inhibitor, whereas the phosphorylate49 kD and 53 kD forms inhibit growth of breast cancer cells. The active53 and/or 49 kD phosphoprotein is expressed by normal human mammarycells, but is not generally produced by human mammary carcinoma cells.Some carcinoma cells make the 44 kD protein that lacks phosphorylationand is inactive.

[0032] The table below summarizes data showing expression and activityof Mammastatin in normal and cancerous cells and tissues.

[0033] Dose response studies with human mammary carcinoma cellsindicates that carcinoma cell growth is 50-70% inhibited with 10 ng/mlof Mammastatin and blocked completely with 25-50 ng/ml. Highlymetastatic cells such as MDA-MB-435 and MDA-MB-231 required 50 ng/ml tostop growth. In vitro and in vivo clinical data experiments indicate theeffect is reversible, and that repeated administration of the inhibitoris required to arrest carcinoma cell growth at the lower concentrations.At doses above 50 ng/ml, however, Mammastatin appears to induceapoptosis, as indicated by histology, e.g. cell necrosis.

[0034] Since Mammastatin is a natural growth inhibitor that blocksmammary carcinoma cell growth, and since no tumors make activeMammastatin, Mammastatin replacement therapy is ideal for therapeutictreatment of breast cancer. The clinical data provided in the examplesbelow demonstrate the effectiveness of Mammastatin replacement therapy.

[0035] The nucleic acid sequence encoding Mammastatin protein has nowbeen isolated, characterized, sequenced (Sequence ID NO:1), determinedto encode all three (53, 49, and 44 kD) molecular weight proteins, andgiven the name “Mammastatin”. Differences in the molecular weight of thethree forms has been determined to be caused by the extent of theprotein's phosphorylation. Mammastatin produced by normal human mammarycells (NHMC) in culture and recombinantly expressed Mammastatin inhibitthe growth of human mammary carcinoma cells, and is useful as atherapeutic agent in the treatment of breast cancer.

[0036] Analysis of human sera from normal women and from breast cancerpatients indicates that decreased blood levels of Mammastatin correlatewith advancing breast cancer. Screening and monitoring blood serum forthe presence of this active inhibitor as described in the examples belowprovides a specific and effective diagnostic tool.

Nucleic Acid Sequence

[0037] The Mammastatin DNA nucleic acid (SEQ ID NO:1) is shown in thetable below, and was identified by cloning and sequencing of MammastatincDNA from a normal human mammary cell cDNA library, as described morefully in the Examples below. Chromatographically purified inhibitor hadnot previously been sufficiently isolated to permit its amino acidanalysis, and early attempts to sequence the protein inhibitor bystandard techniques failed. Attempts to screen a cDNA library usingantibodies raised against chromatographically purified inhibitor proteinfailed to generate an active clone. To overcome these problems, the geneencoding Mammastatin was identified by peptide sequencing and degenerateoligonucleotide screening of a normal human mammary cell cDNA library.

[0038] Concentrated protein produced by normal human mammary cells wasaffinity purified using an anti-Mammastatin antibody raised againstchromatographically purified inhibitor. Purified protein fractions weresupplemented with a small amount (10⁵ cpm) of ³²P labeled as tracers.The labeled tracer protein was purified from conditioned media of cellsgrown in the presence of ³⁵P, as described more fully in the examplesbelow. The protein was cleaved with cyanogan bromide, and cleavedfragments were identified as Mammastatin by autoradiographic analysis of³²P-labeled protein. The most abundant labeled peptides generated by thecleavage were sequenced.

[0039] Two peptides, selected as having unique amino acid sequences(seq. ID Nos. 2 and 3), were used to produce degenerateoligonucleotides. The degenerate oligonucleotides were then used toscreen a normal human mammary cell cDNA library.

[0040] One clone, labeled pMammA, hybridized to oligonucleotides fromboth selected peptides. This clone was further characterized, and wasshown to express protein recognized by anti-Mammastatin antibodies. Theclone has been verified as encoding Mammastatin by Northern blotanalysis, in vitro transcription and translation assays, and growthinhibition assays. A pcDNA3 clone containing the Mammastatin cDNA insert(pMammB) was deposited with the American Type Culture Collection andgiven Accession Number 97451. The recombinant protein expressed frompMammB (Sequence ID No: 2) has been detected by immunoblot oftransfected mammalian cell lines and has been demonstrated to possessgrowth inhibitory activities against mammary cancer cells. The cDNAclone has been completely sequenced (see Example 3) and found to beunique to the BLAST DNA database.

[0041] The nucleic acid sequence of the invention (Sequence ID No: 1)encodes human Mammastatin, which functions to inhibit the growth ofhuman mammary cells, normal and cancerous. The term “human” is notintended to limit the source of the protein nor to limit its inhibitoryeffects only to human cells and tissues. It is understood that thenucleic acid sequence and amino acid sequence of Mammastatin inindividuals may vary somewhat, without altering the structure orfunction of the protein. Further, one skilled in biochemistry willappreciate that modifications of the nucleic acid or amino acid sequencemay be made without altering the structure and/or function of themolecule. For example, the nucleic acid sequence may be modified topermit optimal expression of the desired amino acid sequence using knownoptimal codons for a particular cellular host.

[0042] The nucleic acid sequence of the invention is useful in producinglarge quantities of highly purified Mammastatin protein for use intherapeutic and diagnostic methods in the treatment of breast cancer.

Anti-Mammastatin Antibodies

[0043] Several anti-Mammastatin antibodies have been produced andcharacterized. See, for example, PCT application WO 89/11491 publishedNov. 30, 1989. These antibodies were raised against chromatographicallypurified inhibitor protein, and have been demonstrated to block theinhibitory effect of Mammastatin protein on mammary cell growth.

[0044] Available anti-Mammastatin antibodies include 7G6 and 3C6,commercially available from Neomarkers (Freemont, Calif.) and 6B8.Hybridoma cells producing 6B8 antibody are available from the AmericanType Culture Collection (ATCC No. HB 10152). Each of these antibodiesbinds to all three molecular weight forms of Mammastatin and are usefulin immunological assays, including dot blots and Western blots. The 7G6antibody is preferred for Western blot analysis or for ELISA analysis ofdenatured protein samples. The antibodies 3G6 and 6B8 may be used inELISA assays, e.g., under conditions specified in the examples.

[0045] Additional antibodies can be produced using standard methodsknown for producing monoclonal or polyclonal antibodies. The antigenused to produce antibodies may be derived from culture of NHMC or fromrecombinantly expressed Mammastatin.

Diagnostic Method

[0046] The invention further provides an in vitro assay for detectingactive, inhibitory Mammastatin in patient samples, including tissues,cells, and fluids. Breast cancer disease and advancing metastaticdisease is diagnosed by correlating the presence and type of Mammastatinprotein in a patient's sample with that of normal or cancerous humanmammary cells. A patient's blood or tissue sample is analyzed forMammastatin protein, e.g., for the abundance of Mammastatin proteinand/or for the molecular weight forms of Mammastatin. As discussedbelow, the absence or loss of Mammastatin, particularly of the highermolecular weight, phosphorylated forms of Mammastatin, is correlatedwith breast cancer and indicative of advancing metastatic disease.

[0047] Analysis of Mammastatin is preferably by immunoassay, includingELISA or Western Blot analysis of a patient's blood samples, usinganti-Mammastatin antibodies. Preferably, recombinant Mammastatinstandards are used to provide a standard curve for reliable quantitationof inhibitor levels. Such immunoassays are exemplified by the dot-blotassays and Western blot assays shown in the examples below. In analternative preferred embodiment of the invention, tissue samples, suchas tumor biopsies, are analyzed by immunohistochemistry, or by culturinga patient's tumor cells and examining the cultures for expression ofMammastatin.

[0048] In a particularly preferred embodiment, an assay for thediagnosis of breast cancer includes at least two specific antibodies: anantibody to identify the sampled breast tissue as epithelial tissue,such as an anti-cytokeratin antibody, and an anti-Mammastatin antibody.For example, using an immunoblot format, tissue suspected of containingbreast cancer cells is homogenized, separated on an SDS/PAGE gel,transferred to membrane, and probed with both anti-keratin andanti-Mammastatin antibodies. Isotype specific second antibodies that areconjugated to a suitable marker system such as peroxidase or alkalinephosphatase are used to detect bound antibodies. Membranes containingbound first and second antibodies are then developed using knowncolorometric or fluorometric techniques and quantitated by knownmethods.

[0049] In the most preferred embodiment, the sample is analyzed for thephosphorylated forms of Mammastatin, such as by Western Blot, usinganti-Mammastatin antibodies. A decline or absence of the high molecularweight (53/49 kD) Mammastatin correlates with advancing breast cancer.

Recombinant Expression Vectors and Transformed Cells

[0050] Recombinant expression vectors of the invention are useful forproduction and amplification of purified Mammastatin protein andportions thereof, and for easy isolation of Mammastatin protein andportions thereof to be used in diagnostic and therapeutic methods. Atarget sequence, such as all or a portion of the 2.434 kb MammastatincDNA (SEQ ID NO: 1), is cloned into a suitable nucleic acid sequenceexpression vector such as pUC18, pKC30, pBR322, pKK177-3, pET-3, pcDNA3(In Vitrogen) for COS and CHO cells, and pAcG3X baculovirus expressionvector (PharMingin, San Diego, Calif.) for expression in insect cells,and like, known expression systems by standard methods. Commerciallyavailable expression vectors provide for cloning of a target sequenceinto a site of the vector such that the target sequence is operablylinked to transcriptional and translational control regions.

[0051] The expression vector is then introduced into suitable host cellsusing known methods such as calcium phosphate precipitation, liposomemediated transformation, protoplast transformation, electroporation, andthe like. Suitable host cells include COS and CHO cells, High 5 and SF9insect cells, baclovirus, and yeast cells. Other host cells include E.coli strains such as E. coli DH5α, and avirulent isogenic Salmonellaspp. such as S. typhimurium deletion mutants lacking adenylate cyclaseand cAMP receptor protein, Salmonella mutants in aro genes, and otherSalmonella vaccine strains as described in Bio/Tech, 6:693 (1988).

[0052] Preferably, the cellular host is a Eukaryotic cell, capable ofexpressing the protein with proper folding and kinase activity toproduce a phosphorylated, active inhibitor. Host cells may be screenedby transfection with cDNA encoding Mammastatin. Analysis of the proteinproduced by the transformed cells, e.g. by immunoblot, and the abilityof the protein to inhibit mammary cell growth, for example MCF7 cellgrowth, as described in the examples recited below, can be used toscreen potential host cell systems.

[0053] Host cells transformed with the target nucleic acid sequence arescreened by a variety of methods including colony hybridization orreactivity with antibodies specific for Mammastatin protein. Atransformed cell is a suitable host cell carrying a pcDNA3 or otherplasmid or vector containing a nucleic acid sequence encodingMammastatin. One such plasmid is the pcDNA plasmid (pMammB) carrying the2.4 kb BamHI-XhoI insert from pMammA, deposited with the American TypeCulture Collection in Rockville, Md. on Feb. 22, 1996, and was givenAccession No. 97451. (See Example 5.)

[0054] An expression vector containing the specific target DNA sequenceis used to generate all or a portion of Mammastatin protein, by in vitrotranscription and translation by insertion into cellular hosts forprotein production. Proteins produced from the expression vector systeminhibit the growth of mammary cells, normal and cancerous. (See Example7.) Eucaryotic cells, e.g., Cos7 host cells, transfected with the vectorexpress and secrete Mammastatin into the conditioned medium. Conditionedmedium inhibited the growth of normal and cancerous mammary cells. (SeeExample 8.)

Amino Acid Sequence

[0055] The Mammastatin protein (Sequence ID No. 2) is a polypeptide ofabout 2400 amino acid residues having the sequence deduced from thenucleic acid sequence (SEQ. ID NO: 1) and shown in Table 1. Proteinsynthesized from the cloned Mammastatin nucleic acid sequence (Seq. IDNo. 1) inhibits of breast cancer cell (MCF-7) growth.

[0056] Recombinant Mammastatin protein can be efficiently produced inpurified form and in large quantities. Purified recombinant Mammastatinis useful as a reliable standard for diagnostic assays of the inhibitorin patient samples. Recombinant Mammastatin protein is also useful as apurified therapeutic agent to inhibit or prevent the growth of breastcancer cells.

Therapeutic Use

[0057] Mammastatin protein for therapeutic use is produced from NHMCcultures under serum free conditions or by recombinant means.Mammastatin phosphoprotein is used therapeutically to inhibit mammarycell growth, e.g., in the treatment of breast cancer. Preferably,Mammastatin is produced in higher eucaryotic cells to achievephosphorylation of the protein. Recombinant Mammastatin protein isproduced in host cells or by synthetic means.

[0058] Functional Mammastatin is administered to patients by knownmethods, for the administration of phosphoprotein, preferably byinjection, to increase inhibitor levels in the bloodstream and increasethe inhibitor's interactions with mammary cells.

[0059] The protein may be delivered to the patient by methods known inthe field for delivery of phosphorylated protein therapeutic agents. Ingeneral, the inhibitor is mixed with a delivery vehicle and administeredby injection.

[0060] The dosage of inhibitor to be administered may be determined byone skilled in the art, and will vary with the type of treatmentmodality and extent of disease. Since Mammastatin inhibits approximately50% of mammary cancer cell growth at a concentration of 10 ng/ml andstops growth at about 20-25 ng/ml in vitro, a useful therapeutic dosagerange is about 2.5 ug to about 250 ug administered daily dose. Preferredis approximately 125 ug daily administered dose. The aim of theadministration is to result in a final body dose that is in thephysiological or slightly higer range (50-75 ng/ml). Higher doses ofinhibitor (>50 ng/ml) appear to induce apoptosis, as seen in histologyof treated cells. For clinical use, the preferred dosage range is about500 ng/ml for initial treatment of metastatic disease, followed by amaintenance dosage of about 50 ng/ml. Initial clinical studies, reportedin the examples below, indicate an administered daily dose of about 50ng/ml to about 750 ng/ml is sufficient to induce remission in Stage IVbreast cancer patients.

[0061] Since active Mammastatin is a phosphorylated protein, it isanticipated that multiple doses of the inhibitor will be required tomaintain growth inhibiting levels of Mammastatin in the patient's blood.Also, since Mammastatin generally acts as a cytostatic agent rather thana cytocidal agent, it is expected that a maximum effect of the inhibitorwill require regular maintenance of inhibitor levels in breast cancerpatients.

[0062] In its preferred use, Mammastatin is administered in high dosages(>50 ng/ml, preferably about 50-500 ng/ml) to induce tumor regression.Lower, maintenance doses (<50 ng/ml, preferably 20-50 ng/ml) are used toprevent cancer cell growth.

[0063] Clinical experience with administered Mammastatin in Stage IVbreast cancer patients indicates a useful dose is that which maintainsphysiological levels of Mammastatin in the blood. Administration ispreferably daily, but, may be, for example, by continuous infusion, byslow release depot, or by injection once every 2-3 days. Anecdotalevidence suggests continuous administration may induce feedbackinhibition, thus, a preferred administration scheme is to administerdaily dose of Mammastatin for approximately 25-28 days, followed by 2-5days without administration.

Diagnostic Use

[0064] Assays of the present invention for detecting the presence of thefunctional inhibitor in human tissue and serum are useful in screeningpatients for breast cancer, for screening the population for those athigh risk of developing breast cancer, for detecting early onset ofbreast cancer, and for monitoring patient levels of inhibitor duringtreatment. For example, analysis of a patient's blood Mammastatin mayindicate a reduced amount of high molecular weight, phosphorylatedMammastatin, as compared with a normal control or with the patient'sprior Mammastatin profile. Such a change is correlated with increasedrisk of breast cancer, with early onset of breast cancer, and withadvancing metastatic breast cancer. Diagnostic assay for phosphorylated,active, 49/53 kD Mammastatin preferably is by Western blot immunoassay,e.g. ELISA, or using specific anti-Mammastatin antibodies. Screening,for example, in serum, is preferably by immunoassay, e.g., dot blotassay.

[0065] For best results, the patient samples should be assayed within ashort time of sampling (within one week), stored at 4° C. (less than oneyear), or frozen for long term storage. Most preferably, samples arefrozen until time of assay.

Assay Kit

[0066] In a specific embodiment of the invention, an assay kit for thedetection of Mammastatin in a patient's fluid and/or breast tissue isprovided. The preferred screening assay is an immunoassay such as a dotblot assay to detect or quantitate Mammastatin in blood serum. Such ascreening kit includes anti-Mammastatin antibodies and optionally acontrol antibody and/or Mammastatin controls or standards. A secondscreening assay analyzes Mammastatin in breast tissue. Preferably, theassay kit contains necessary reagents and tools for reacting the tissuewith an antibody to specifically determine that the tissue is breastepithelium, e.g., an anti-cytokeratin antibody, and a specificanti-Mammastatin antibody. The commercially available antibody mixture,pan-keratin (Sigma) is a preferred anti-cytokeratin antibody.

[0067] A negative assay for Mammastatin could be caused by either thepresence of a breast cancer tumor, or by non-epithelial breast tissue.Use of the anti-cytokeratin antibody guards against false positiveassays. Epithelial cells of the breast that do not stain with theanti-Mammastatin antibody or which only express the 44 kD Mammastatinare transformed cells. Thus, by first identifying the tissue as breastepithelium, e.g., isolated from breast tissue and positive with theanti-cytokeratin antibody, and then identifying a second positivereaction with anti-Mammastatin antibody, false positives are avoided.

[0068] Because about 30% of the breast cancer cells studied to dateexpress non-phosphorylated inactive, 44 kD Mammastatin, the preferredmethod of analysis is to differentiate between the 53/49 kD and 44 kDforms, e.g. by Western blot analysis.

[0069] The invention is further defined by reference to the followingexamples:

EXAMPLE 1 Human Mammary Cell cDNA Library

[0070] A cDNA library was prepared from human mammary cells obtainedfrom reduction mammoplasties (UM Hospital). Total RNA was isolated fromthe mammary cells by cesium chloride gradient. From the total RNApreparation, mRNA was isolated. The methods used were those described inGarner I., “Isolation of total and poly A+ RNA from animal cells”,Methods Mol. Biol. (1994) 28:41-7.

[0071] Reverse transcriptase in the presence of the isolated mRNAproduced cDNA that was then ligated to EcoRI linkers. The cDNA wasinserted into EcoR1 cut T4 DNA ligase-treated Lambda Zap, and amplifiedin XL1-blue E. coli, following the method described in Short J M., etal. (1988) Nucleic Acids Research 16: 7583.

EXAMPLE 2 Preparation of Mammastatin Oligonucleotides

[0072] The normal human mammary cell cDNA library prepared in Example 1was screened for the presence of nucleic acids encoding Mammastatinusing degenerate oligonucleotides. The degenerate oligonucleotides werederived as follows:

[0073] Normal human mammary cells were obtained from the Plastic SurgeryDepartment of the University of Michigan Hospital or from theCooperative Human Tissue Network. The tissue was reduced by collagenasetreatment generally following the procedure described in Soule, et al.,In Vitro, 22:6 (1986).

[0074] Mammary cells were grown to confluence in 175 cm² flasks inDMEM/F12 low calcium media formulated with 40 μM CaCl₂ and supplementedwith 5% CHELEX treated equine serum (Sigma), 0.1 μg/ml cholera toxin(Sigma), 0.5 μg/ml hydrocortisone (Sigma), 10 ng/ml epidermal growthfactor (EGF, Collaborative Research, Bedford Mass., 10 μg/ml insulin,and 1 μg/ml penicillin/streptomycin following the method described inSoule, et al., In vitro 22:6(1986). Equine serum was treated with CHELEXresin for three hours at room temperature to remove serum calcium.

[0075] Cell lysates were prepared by rinsing cells with TBS and scrapingfrom the flask with a Teflon scraper. Cells were collected bycentrifugation and lysed with 8M Urea, 50 mM TRIS pH 7.5, 0.5%Beta-mercaptoethanol, 0.5% TRITON X-100 (lysis buffer) and three minutesof sonication on ice.

[0076] The cell lysates were fractionated on DEAE-Sephacel anionexchange resin (Sigma) equilibrated with lysis buffer. Lysates wereloaded onto the resin filled columns (50 ml disposable, Bio Rad) andwashed with ten column volumes of the lysis buffer. Material flowedthrough the columns with only gravity feed. Fractions were eluted with asalt gradient produced by continuous gravity feed of elution buffercontaining 5M NaCl into a closed mixing chamber initially containingelution buffer (250 ml of 8M urea and 50 mM TRIS pH 7.5) in the absenceof salt.

[0077] Elution fractions (2 ml) were collected with a Gibson fractioncollector, and were analyzed for the presence of mammary cell growthinhibitor by dot blot with the anti-Mammastatin antibody, 7G6, describedabove.

[0078] Positive fractions were pooled and dialyzed into lysis bufferwith 50 mM NaCl, and were again separated on an identical ion exchangecolumn and eluted with a continuous decreasing pH gradient (pH 8 to pH3)in elution buffer with 50 mM NaCl. (To produce the pH gradient, pH3buffered urea was continuously mixed with the initial pH8 buffer.)Fractions (2 ml) were collected and analyzed with the 7G6 antibody asdescribed above.

[0079] Positive fractions were again pooled and concentrated to{fraction (1/10)} the original volume by filtered centrifugation (AmiconCentriprep, 10 kD cutoff). The concentrated pool was size fractionatedby preparative SDS polyacrylamide gel electrophoresis (PAGE) along withprestained molecular weight standards (Sigma).

[0080] Protein contained in the molecular weight range between 40 and 60kD was excised from the gel in 0.5 cm strips or fractions.Electroelution of the protein from each gel strip was carried out byplacing the gel strip in 1 ml of running buffer (192 mM glycine, 25 mMTRIS pH 8.3, 0.1% SDS) in dialysis tubing. The tubing was placed in asubmarine electrophoresis apparatus and electroeluted overnight at 25volts. Current was reversed for 2 minutes and running buffer, nowcontaining the electroeluted protein, was removed. Purity of the elutedprotein was checked by analytic SDS PAGE with silver-staining, and alsoby immunoblot with the 7G6 antibody, following the procedure describedin Towbin et al., J. Clin. Chem. Clin. Biochem. 27:495-501 (1989).Fractions that were at least 70% pure as determined by silver-stainedPAGE were pooled, concentrated, and lyophilized to powder form.

[0081] The pooled protein was cleaved with cyanogen bromide byresuspending lyophilized powder in 500 μl of 70% formic acid andincubating overnight at room temperature (about 20 hours) with 20 mg/mlof cyanogen bromide (Sigma). The methods used are described in Freemont,et al., Arch. Biochem. Biophys. 228:342-352 (1986). Cyanogenbromide-cleaved protein samples were dialyzed into double distilled,deionized water and again concentrated and lyophilized to powder.

[0082] Cyanogen bromide cleavage generated multiple peptides from theoriginal protein sample, which were separated by preparative 15% SDSPAGE and transferred onto PVDF membrane by electroelution.

[0083] In addition to the protein obtained from mammary cell lysates,protein was also isolated from normal human mammary cell conditionedmedium. Normal cells were incubated with 8 ml DMEM lacking phosphatesand supplemented with 200 μCi/ml ³²P-ortho-phosphate and 1% dialyzedfetal bovine sera. Cells were allowed to grow for 24 hours in thepresence of the ³²P before conditioned media was collected.

[0084] The collected conditioned media was concentrated 5× by Amiconfiltration with 10 kD exclusion limit. Concentrated media was rinsedonce with PBS on filtration membranes to remove excess unincorporatedphosphate and was further fractionated by S-200 SEPHACRYL (Pharmacia,Upsala, Sweden) molecular sieve chromatography (100 cm×0.75 cm column)eluted with PBS. Both the filter and the column permit removal ofunincorporated ³²P from the sample. One ml fractions were collected fromthe column, and labeled fractions identified by scintillation counting.Radioactive fractions were pooled and analyzed by SDS PAGE with silverstaining and autoradiography. The pooled protein was concentrated,lyophilized to powder, and combined with the larger mass of unlabeledprotein purified as described above, before cyanogen bromide cleavage.The addition of labeled protein provided a convenient means of tracingcyanogen bromide cleavage fragments containing phosphorylatedMammastatin peptides. Cleaved peptides were separated on preparativePAGE as described above.

[0085] After radioactive proteins were cyanogen bromide cleaved,separated, transferred to PVDF membrane, and exposed to Xray film, twolabeled bands of approximately 20 and 22 kD were seen. These twopeptides were excised from membranes and sequenced by Edman degradationmethods at the University of Michigan Biomedical Research Core Facilityusing methods described in Ullah Alt et. all., Biochem. Biophys. Res.Comm. 203:182-189 (1994). The amino acid sequences of each of the twopeptides was compared with known database sequences using the NIH“BLAST” server. The two peptides appeared to be unique.

[0086] A particularly unique portion of each sequence was used toproduce degenerate oligonucleotides, using the standard third positiondegeneracy according to the method described in Jerala, Biotechniques13:564-567 (1992). From the 20 kD peptide, the sequence“gly-gln-leu-glu-tyr-gln-asp-leu-arg” (Seq ID No. 3) was used; from the22 kD peptide, the sequence“tyr-glu-arg-asp-leu-lys-gly-arg-asp-pro-val-ala-ala” (Seq ID No. 4) wasused to generate multiple species of oligonucleotides. The degenerateoligonucleotides were purified by high pressure liquid chromatography.!SEQ. ID NO.? Peptide 3 gly gln leu gle tyr gen asp leu arg 4 tyr gluarg asp leu lgs gly arg asp pro val ala ala

[0087] The degenerate oligonucleotides were end-labeled with ³²P-gammaATP and T4 DNA polynucleotide kinase (BRL, Bethesda, Md.) andresuspended in T4 DNA kinase buffer (60 mM TRIS pH 7.8, 10 mM MgCl₂, 15mM beta-mercaptoethanol) at 1.5 mg/ml. Oligonucleotides (250 μM) werethen incubated with 0.33 μM ATP, 5 units kinase in 25 μl kinase buffer,for two hours at 37° C. Incorporation of ³²P-phosphate was determined byTCA precipitation (15% TCA, 4° C., 15 minutes). Typical incorporationwas 10⁹ cpm/μg DNA.

EXAMPLE 3 Screening Mammary Cell cDNA Library with DegenerateOligonucleotides

[0088] Bacteria infected with phage prepared for Example 1, containing anormal mammary cell cDNA insert, were plated on 15 cm NZCYM (10 g, NZamine (Bohringer Manheim), 5 g NaCl, 5 g yeast extract, 2 g MgSO₄, 1 gcasamino acids) plates in top agar (1/10 dilution of infected bacterialcultures to 6 ml of 7% NZYM top agar) and allowed to incubate eighthours at 37° C. Plates containing plaques were overlaid withnitrocellulose for 15 minutes before denaturation of phage. Phage wasdenatured by blotting filters (DNA side up) on Whatman paper saturatedwith 0.5 M NaOH, 1.5 M NaCl for 5 minutes. Filters were rinsed with H₂Obefore incubating for 5 minutes in 1 M TRIS pH 7.0, 1.5 M NaCl followedby 20× SSC and 2× SSC, each for 5 minutes. Filters were dried and bakedfor 1 hour at 80° C. or placed under ultraviolet light to immobilizeDNA. Baked filters were washed for 30 minutes in 2× SSC with 1% SDS andthen prehybridized with 50% deionized formamide, 5× Denhart's solution,1% SDS, 5× SSC and 100 μg/ml sheared salmon sperm DNA overnight at 37°C.

[0089] Filters were hybridized with the labeled degenerateoligonucleotide prepared as described for Example 2 in prehybridizationbuffer to which 10⁷ cpm/ml of heat-denatured (95° C., 5 minutes) labeleddegenerate oligonucleotide had been added. Hybridizations were performedat 37° C. for 24 hours. Filters were washed with 2× SSC for thirtyminutes at 37° C. followed by 3 washes in 2× SSC plus 1% SDS at 50° C.for thirty minutes. Filters were rinsed with 2× SSC briefly, dried andexposed to Kodak AR-5 filn for 24-48 hours to identify positive plaques.

[0090] Positive plaques were isolated from agar plugs excised using areversed 200 μl sterile pipette tip, and resuspended in SM bufferovernight at 4° C. Secondary and tertiary plates (10 cm) were made usingXL1-B infected with 1/10,000 dilution of phage containing SM buffer, tobacteria, in NZCYM (with 1 mM MgSO4). Plaques were produced byincubating infected bacteria for 8 hours as described above, and werethen transferred to nitrocellulose before screening with labeleddegenerate oligonucleotides. Screening was performed essentially asdescribed in Kroczek R A., J Chromatogr 618:133-45(1993), using 10⁷cpm/ml of labeled DNA for hybridizations and a final wash stringency of2×SSC at 50° C. for thirty minutes.

[0091] The clone selected for further analysis was one recognized byboth of the degenerate oligonucleotides. This clone was given the name“pMammA”.

EXAMPLE 4 Sequencing of Mammastatin cDNA

[0092] The positive clone obtained in Example 3, pMammA, was sequencedby an automated sequencer at the Biomedical Research Core Facility atthe University of Michigan and also by dideoxy DNA sequencing using 15%DNA sequencing gels and radiolabeling the DNA fragments with ³⁵Snucleotides. The methods used are described in Lasken R S., et al. ProcNatl Acad Sci USA 82:1301-5(1985). The nucleic acid sequence obtained isshown below in Table 1 (Seq ID No. 1).

[0093] The recognized error rate of automatic sequences is about 5%.Therefore, the clone deposited is resequenced for confirmation of thenucleotide sequence, particularly mindful of areas suspected ofpotential errors, as noted.

EXAMPLE 5 Subcloning the Mammastatin cDNA into an Expression Vector

[0094] The Mammastatin cDNA insert, pMammA, was subcloned into theexpression vector, pcDNA 3 (In Vitrogen). The Mammastatin cDNA wasisolated by digesting the pMammA plasmid obtained as described forExample 4 with BamH1 and Xho1 restriction endonucleases. The restrictionenzymes cut the plasmid at the ends of the Mammastatin clone insert,creating a linear plasmid fragment and a linear insert fragment. Thedigested sample was placed in the wells of a 1.2% agarose gel submergedin an electrophoresis apparatus, a 50V current was applied for twohours. Electrophoresis separates DNA fragments on the basis of size withthe larger plasmid DNA fragment having the slower migration rate on thegel. The portion of the agarose gel containing the 2.4 kb was visualizedby ethidium bromide staining and observing the gel over an ultra-violetlight box. The 2.4 kb Mammastatin fragment was cut from the gel andplaced into dialysis tubing and the DNA was electroeluted intotris-borate buffer, TBE: (0.089M Tris-borate, 0.089M boric acid, 0.002MEDTA) that was collected and precipitated with ethanol.

[0095] The pcDNA3 plasmid DNA was modified to accept the MammastatincDNA fragment during ligation. pcDNA3 plasmid was digested with BamH1and Xho1 restriction endonucleases and after digestion was complete, theDNA was incubated for one hour in the presence of calf intestinalphosphatase to remove 5′ phosphates. The pcDNA3 sample was then phenolextracted and ethanol precipitated.

[0096] The pcDNA3 and the Mammastatin 2.4 kB cDNA fragment were ligatedtogether. The 2.4 kb Mammastatin fragment and the linear pcDNA3 plasmidwere mixed in a 3:1 ratio in the presence of T4 DNA ligase. The ligationreaction was allowed to incubate for one hour and then stored at 4° C.overnight. After the ligation reaction was completed the DNA was used totransform E. coli competent cells. Subcloning was verified by purifyingplasmid DNA from ampicillin selected colonies. The plasmids weredigested with the restriction endonucleases BamH1 and XhoI. The digestedDNA samples were placed in an agarose gel and separated byelectrophoresis. A plasmid containing the correct size Mammastatin DNAfragment was designated pMammB, and was deposited with the American TypeCulture Collection (ATCC) on Feb. 22, 1996, and given accession number:ATCC 97451.

EXAMPLE 6 Transfection and Protein Expression from the Mammastatin cDNASequence

[0097] Cos-7 cells do not express immunoreactive proteins thatco-migrate with the Mammastatin proteins. pMammB and PCDNA3 were used totransfect Cos-7 monkey fibroblast cells using LIPOFECTIN® (BRL, LifeTechnologies, Bethesda, Md.) using the manufacturers suggested protocol.The transfected cells were grown for two days prior to harvest.Transfected cells were removed from plates by trypsinizaton of cellsusing standard protocols. (2.5 ml's of Trypsin (0.25% SIGMA) wasincubated in flasks of cells at 37° C. for 5 minutes. A 7.5 ml aliquotof RPMI media with 10% FBS (fetal borin serum) was added and cells werecollected by centrifugation.) Cells were counted by hemocytometer andlysed in SDS PAGE sample loading buffer at 10⁷ cells/ml. Cell lysateswere separated on 8-15% SDS-PAGE gradient gels (Biorad) and transferredto a nylon membrane using methods described in Towbin H., et al., J.Clin Chem Clin Biochem (August 1989) 27(8):495-501. The membrane wasprobed with anti-Mammastatin monoclonal antibody 7G6. Bound antibody wasdetected with peroxidase conjugated GAM-IgM and developed by ECL(Amersham).

[0098] As shown in FIG. 1, Cos-7 cells transfected with pMammB (lanesC,D) expressed immunoreactive proteins that co-migrated with Mammastatinprotein (lane A). Cos-7 cells transfected with the empty vector PCDNA3alone did not express immunoreactive proteins when immunoblotexperiments were performed (lane B). DNA/AA SEQUENCE Lane A NHMC (25μg) - control Lane B Cos pcDNA3 cell lysate (25 μg) - control Lane CCos-pMammB cell lysate (10 μg) Lane D Cos-pMammB cell lysate (20 μg)

[0099] The immunoblot experiments illustrate the pMammB clone contains acDNA insert capable of synthesizing a protein with the size andimmunologic characteristics of Mammastatin. In addition, immunoreactiveproteins of 44, 49 and 53 kD were expressed in Cos-7 cells transfectedwith pMammB. These proteins migrated at the same molecular weight as theMammastatin proteins previously identified in normal human mammarycells. This group of immunoreactive proteins was not identified in Cos-7cells transfected with the empty vector, pcDNA3.

[0100] In the particular assay shown in FIG. 1, the NHMC control showsan unusually high amount of 44 kD Mammastatin. This is an artifactproduced by long term (>1 yr) storage of the NHMC standard at 4° C.,causing degradation of the higher molecular weight forms, over time.When fresher NHMC samples (<1 yr old) or frozen samples are used, the 44kD protein is always less abundant than the higher molecular weightforms.

EXAMPLE 7 GST Fusion

[0101] The Mammastatin clone can be similarly subcloned into abaculovirus expression system. The pMammA insert has been subcloned intoa pAcG3× vector obtained commercially from Pharmgen (San Diego, Calif.).This vector allows production of Mammastatin as a fusion protein withglutathione S-transferase (GST), having a portion of the GST geneupstream of the coding site.

[0102] The pMammA insert was subcloned by preparing sets of PCR primersthat contained Bam H1 (5′) and Sma 1 (3′) restriction enzyme recognitionsites, a small, non-specific region, and a portion of the Mammastatinsequence. Three sets of primers, each shifted in reading frame, wereprepared. The primers hybridized to the pMammA clones and in a typicalPCR reaction with pMammA template DNA, amplified a pMammA PCR productcapable of insertion into the reading frame of the GST gene in pAcG3X.The vector was then used to transfect High 5 (In Vitrogen) host insectcells, and express a GST-Mammastatin fusion protein that was easilypurified from host insect cells using glutathione resin (glutathioneagarose, Qiagen, Chatsworth, Calif.).

[0103] To prepare DNA for insertion into the BamH1, Sma 1 restrictionsite of pAcG3X (PharMingen, San Diego, Calif.), primer sets wereprepared in three reading frames to include, for the 5′ primer, theBamH1 recognition site (GGATCC), a portion of the pMammA sequence, andsome 5′ sequence from the pBluescript vector. The 3′ primers wereidentical, and included the Sma1 recognition sequence (GGG CCC), aportion of the pMammA sequence, and some pBluescript sequence.

[0104] The primer sets used are shown in the following table: Seq. IDNo: 5′ Primers (in three reading frames)* 5 5′- TGG GAT CCC TTC GCC ACGAGC ACG GTG -3′ 6 5′- TGG GAT CCT TCG CCA CGA GCA CGG -3′ 7 5′-TGG GAT CCC CTT CGC CAC GAG CAC -3′ 3′Primer 8 5′- TTT TTT TTT TTTGGG CCC TTA AGT 3′**

[0105] Only one primer set (Seq. ID NOS. 6 and 8) produced clonescapable of coding for active inhibitory Mammastatin. The active clones,when used to transform High 5 cells, produced Mammastatin that wasimmunologically reactive in the transformed cells (see FIG. 2).

[0106] Other known eukaryotic expression systems may similarly be usedto produce Mammastatin protein.

EXAMPLE8 Inhibition Assay with Proteins Produced by In VitroTranscription and Translation

[0107] In vitro transcription of pMammB, Mammastatin cDNA was performedusing a Stratagene, Express RNA transcription kit to produce MammastatinRNA. The RNA produced was translated into protein using the StratageneIn Vitro Express translation kit. Mammastatin protein produced fromtranslation of the Mammastatin RNA was shown to inhibit mammary cellgrowth in culture.

[0108] Cultures of MCF-7 cells were treated with protein productsproduced in the translation assays described above. Protein products (5%by volume, culture medium) were added to cells in 12-well platescontaining 1 ml medium per well. Parallel cultures were treated withboth the translation product and the anti-Mammastatin antibody 3C6, at30 μg/ml final concentration.

[0109] As a negative control, cultures were treated with proteinproducts translated with the Stragene In vitro Express Translation kitincubated in the absence of Mammastatin cDNA (i.e. employ vector). Theselysates do not have the proper machinery to produce the Mammastatinprotein.

[0110] All cultures were allowed to grow for six days after beingtreated with the protein products and the cell number of each sample wascalculated using a Coulter counter. There were triplicate samples ofeach culture condition so that the cell number of each sample wasaveraged and percent inhibition was determined by comparison to thereticulocyte lysate treated control cells.

[0111] As shown in FIG. 3, the protein translation product of pMammBinhibited MCF-7 cell growth. This inhibition was greatly reduced orblocked in the presence of anti-Mammastatin antibody, 3C6.

EXAMPLE 9 Inhibition of Mammary Cells with Proteins Present withinConditioned Media Obtained from Growing Cos-7 Cells Transfected withpMammB

[0112] Mammary cell growth inhibition experiments were performed usingconditioned media obtained from Cos-7 cells transfected with pMammB asdescribed for Example 6. Mammastatin is a secreted protein and is foundin conditioned media of cells expressing the protein. The growthinhibition caused by conditioned media was blocked by the addition ofanti-Mammastatin antibody.

[0113] MCF-7 cells were plated at 10⁴ cells/ml in MEM supplemented with10% non-essential amino acids and FBS (SIGMA). Cells were allowed toattach overnight and were then supplemented with 10% by volume ofconditioned media (3 day culture) from either: (1) Cos-7 cellstransfected with the empty vector pcDNA (Negative control), (2) Cos-7cells transfected with pMammB (pMammB-Cos), (3) NHMC-conditioned media,or (4) non-conditioned media. Parallel MCF-7 cultures were supplementedwith 30 ug/ml of 3C6 blocking antibody. Treated MCF-7 cells were allowedto grow for six days and were then counted by hemocytometer.

[0114] Inhibition of cell growth was determined by comparing the growthof MCF-7 cells incubated in conditioned media with the growth of MCF-7cells incubated in control, non-conditioned media. Data are shown inFIG. 4, and demonstrate that conditioned media from pMammB-transformedcells inhibited mammary cancer cell growth as efficiently as did normalhuman mammary cell conditioned media. This inhibition was blocked in thepresence of anti-Mammastatin antibody.

EXAMPLE 10 Three Immunologically Reactive Anti-Mammastatin Proteins

[0115] Whole normal human mammary cells (NHMC) and mammary carcinomacells in tissue culture cells were lysed, and cell lysate proteins wereseparated by SDS/PAGE as described above and in Ervin, Paul, 1995,Doctoral dissertation, University of Michigan, Chapter 2. Lysed cellsamples were separated on 10% SDS-PAGE in a Mini-Protean II apparatus(25 μg/sample). Proteins were transferred to nitrocellulose and probedwith the anti-Mammastatin monoclonal antibody 7G6 or IgM controlantibody, alkaline phosphatase conjugated second antibody, goatanti-mouse IgM was utilized with an NBT/BCIP substrate system to detectpositive antibody reactions colorometrically. The data are shown in FIG.5. CARCINOMA CELLS LANE 1 ZR-75-1 LANE 2 MDA MB 435 LANE 3 4MCF-7 LANE 4T47D LANE 5 NHMC-14 positive control LANE 6 NHMC-14 positive controlwith the 38C13 antibody NORMAL CELLS LANE 7 NHMC-17 LANE 8 NHMC-16 LANE9 NHMC-15 LANE 10 NHMC-14 LANE 11 NHMC-6 LANE 12 NHMC-14 positivecontrol

[0116] As shown in FIG. 5, normal human mammary cells expressed adoublet of proteins migrating at 49 and 53 kD that were stronglyrecognized by the anti-Mammastatin monoclonal antibody and a thirdweakly immuno-reactive 44 kD protein. The four tumor cell lines testedexpressed either a 44 kD immuno-reactive protein alone (lanes 1,4) or noimmunoreactive protein at all (lanes 2, 3).

[0117] The above data is representative of experiments performed onnormal cells from 42 different reduction mammoplasty patients over aperiod of several years. Expression of the 44 kD protein in normal cellsand cancer cell lines varied in intensity with each preparation.

EXAMPLE 11 Mammastatin is a Phosphoprotein

[0118] Cellular phosphorylated proteins of mammary cells were labeledwith ³²P by supplementing normal mammary cell cultures with³²P-orthophosphate (200 μCi/ml) for 24 hours. Conditioned media wasconcentrated 5× by Amicom Centrifugation with a 30 kD molecular weightrestriction. Concentrated media was rinsed once with PBS on filtrationmembranes to remove excess unincorporated phosphate and fractionated byS-200 SEPHACRYL (Pharmacia, Upsala, Sweden) molecular sievechromatography (100 cm×0.75 cm column) with PBS elution buffer.Immunoblots were prepared as described above and probed with the 7G6antibody.

[0119] A radiolabeled 53 kD Mammastatin protein was identified inconditioned media by immunoprecipitation. This analysis indicatedMammastatin is a secreted phosphoprotein. Since secreted phosphoproteinsare uncommon, Brefeldin A treatment of cells was utilized to determinewhether Mammastatin was present in conditioned media due to secretion orto cell breakage or leaking. Brefeldin A is a fungal compound thatblocks the secretion of proteins from eukaryotic cells. Brefeldin Ainhibits normal endoplasmic reticulum and golgi function and blocksvesicle formation (Ervin, Paul, 1995, Dissertation, Page 25). Since mostsecreted proteins are liberated from the cell by a process of exocytosisfrom membrane bound vesicles, blocking vesicle formation blockssecretion of many proteins. When NHMC are grown in the presence ofBrefeldin A, phosphorylated Mammastatin is not identified in conditionedmedia.

[0120] To determine the amino acid residues that are phosphorylated inMammastatin protein, radiolabeled 53 kD protein was subjected tophospho-amino acid analysis. NHMC cells were incubated with³²P-orthophosphate for 24 hours. Cell lysates were thenimmunoprecipitated with the anti-Mammastatin antibody 7G6 and purifiedas follows. The 53 kD protein was digested with trypsin and hydrolyzedwith acid. Two dimensional thin layer chromatography was used to analyzethe phosphorylated amino acids of Mammastatin. ³²P-amino acids weremixed with phospho-ser/thr/tyr controls and loaded at the origin (0) ofa 2D TLC plate (20 cm). The samples were separated into two dimensions:1st dimension—pH 1.9 Buffer (50 ml formic acid, 156 ml glacial aceticacid/2000 ml (1794 H₂O), 20 minutes @1.5 K volts; rotate clockwise; 2nddimension—pH 3.5 Buffer (10 ml pyridine, 100 ml's glacial acetic acid:1890 ml H₂O) for 16 minutes @1.3 K volts.

[0121] The TLC plates were stained with ninhydrin and exposed to film.Phospho-amino acid analysis demonstrated the 53 kD Mammastatin proteincontained three types of phosphorylated amino acid residues by comparingautoradiographs to ninhydrin stained phospho-amino acid standards.

[0122] Threonine (Th) was the most abundant phosphorylated amino acidfollowed by serine (S) and Tyrosine (Ty), the least abundantphosphorylated species. However, the relative abundance of phosphoaminoacid residues may not be representative of that in the native protein,since acid hydrolysis can free phosphate from phosphotyrosyl residues.

EXAMPLE 12 One Mammastatin Protein with Varied Phosphorylation

[0123] Cellular phosphorylation of proteins can be modulated byphosphatases and kinases. Mammastatin is differentially phosphorylatedin normal and tumor cell lysates due to differential activities ofMammastatin phosphatases. The effect of phosphatase on Mammastatin inNHMC lysates was examined.

[0124] NHMC were grown to confluence in low calcium media and collectedby scraping into TBS. Cells were washed with TBS and resuspended at 2mg/ml in acetate buffer pH 6.6 with 0.5% Triton X-100. 5 μg/ml of eitherYersinia phosphatase (YOP) (Stuckey, et al., Nature 370:571-5 (1994)) orYersinia phosphatase mutant (MYOP) containing an active site mutationwas used to digest cell lysates for six hours at 37° C. (YOP and MYOPwere gifts from Dr. S. Jack Dixon, University of Michigan, BiochemistryDepartment). As shown in FIG. 6, digestion of normal human mammary celllysates with Yersinia phosphatase (YOP) resulted in a reduced amount of53 kD Mammastatin protein identified by anti-Mammastatin immunoblot(lane A). In contrast, digestion with the Yersinia phosphatase mutant(MYOP, lane B), did not alter identification of the 53 kD Mammastatinprotein. These results indicate identification of the 53 kD Mammastatinprotein by immunoblot is a convenient measure of the state ofphosphorylation of the Mammastatin protein.

[0125] Conditioned medium incubated in the presence of Yersiniaphosphatase (YOP), as described above, was used to treat MCF-7 cells. Aspreviously observed, NHMC conditioned medium inhibits the growth ofMCF-7 cells, and this inhibition is blocked by anti-Mammastatinantibodies. As shown in FIG. 7, treatment of NHMC conditioned mediumwith YOP abrogates this inhibitory activity. As a control, treatment ofNHMC conditioned media with a YOP mutant lacking phosphatase activity(M. YOP) was tested. This mutant had no effect on the inhibitoryactivity of NHMC conditioned media. Immunoprecipitation of theconditioned media with the anti-Mammastatin antibody 7G6 removed theinhibitory activity.

[0126] TCA precipitation indicated that incubation of conditioned mediawith YOP removed about 50% of incorporated phosphate. As shown above,YOP also removed the 53 kD species from NHMC lysates (FIG. 6).

EXAMPLE 13 Phosphorylated Mammastatin Produced by Normal But NotCancerous Mammary Cells

[0127] Normal and transformed mammary cells were labeled with ³²Porthophosphate. Carcinoma cell lines were grown in the media assuggested by the ATCC, with the exception of MCF-7 cells which weregrown in MEM (Celox) supplemented with 10% FBS, non-essential aminoacids, and insulin (10 mg/l). ³²P orthophosphate labeling of cellularproteins was performed in phosphate-free DMEM (ICN) containing 2%dialysed FBS. Cells were incubated 24 hours at 37° C. with 200 μC:/ml of³²P-phosphate. After 48 hours, conditioned media was collected from cellcultures and concentrated 5×. Conditioned media was washed with TBS andconcentrated on Amicon filters with a 10 kD mw cut-off. The cell layerwas scraped (using a Teflon cell scraper) into lysis buffer, 1.5ml/flask (0.5% TritonX-100, 2.01% SDS at deoxycholate) from cell lysatesand conditioned media.

[0128] Mammastatin proteins were immunoprecipitated by adding 5 μg 7G6anti-Mammastatin antibody per 500 μl of 5× concentrated media or celllysate and incubating at room temperature for 1.5 hours. Goat anti-mouseIgM second antibody (5 μg/0.5 ml) was added and the mixture incubated anadditional hour. Protein G PLUS/A agarose® slurry (Oncogene Science) wasadded and the mixture incubated 1.5 hours at room temperature toimmobilize antibody complexes.

[0129] The complexes were washed 6× with lysis buffer, each washfollowed by centrifugation at 3000×g. SDS-PAGE loading buffer (50 μl)was added before the sample was heated to 100° C. for 3 minutes.Supernatants were resolved by SDS-PAGE, transferred to nitrocellulose,and exposed to Kodak X-AR film.

[0130] Phosphate labeling of NHMC proteins and subsequentimmunoprecipitation identified 49 and 53 kD phosphoproteins in NHMC. The49 and 53 kD phosphoproteins were not recognized in carcinoma celllines. Carcinoma cell lines MCF-7, T47D, ZR-75-1 and MDA-MB-435expressed a 44 kD immunoreactive protein, but this protein did not labelwith ³²P-orthophosphate.

[0131] This study indicates more incorporated phosphate with increasingmolecular weight of Mammastatin. Lack of phosphorylation of Mammastatinin transformed cell lines correlates with lack of higher molecularweight forms of the protein and lack of Mammastatin inhibitory activity.

EXAMPLE14 Mammastatin Kinase & Phosphatase

[0132] Flasks of normal or carcinoma cells were grown to 75% confluence.Cell cultures were washed three times with TBS and then scraped into TBSwith a Teflon scraper. Cell suspensions were pelleted at 1000 g bycentrifugation and then resuspended in a small volume of TBS. An aliquotof each type of cell was removed for protein quantitation. Proteinconcentrations were then equalized at 2 mg/ml in lysis buffer (TBS with0.5% Triton X-100 and 5 μg/ml each of aprotinin, leupeptin, and PMSF).Equal masses of normal and tumor cell proteins were mixed and incubatedat 37° C. for three hours. Parallel mixtures of normal and carcinomacell lysates were performed in the presence of 10 nM Orthovanedate(NaVO₄), a phosphatase inhibitor. The mixture was then separated bySDS/PAGE and analyzed by Western Blot using the 7G6 antibody. The dataare shown in FIG. 8. LANE A ZR-75-1 Lysate (30 μg) LANE B NHMC Lysate(30 μg) LANE C NHMC (30 μg) + ZR 75 (30 μg) + 10 nM NaVO₄ LANE D NHMC(30 μg) + ZR 75 (30 μg)

[0133] As shown in FIG. 8, cancer cells (ZR-75-1) (lane A) did notproduce 53/49 kD Mammastatin, as compared with NHMC (lane B). Mixing ofnormal and cancer cell proteins, in the presence of proteinases, reducesthe amount of active, 53 kD inhibitor (lane D). However, in the presenceof the tyrosine-phosphatase inhibitor NaVO₄, the 53 kD species isretained in the mix (lane C). These results indicate that carcinomacells express phosphatase activity capable of eliminating phosphorylatedforms of Mammastatin.

[0134] Expression of Mammastatin in normal and transformed cell linescan be measured quantitatively by Western blot analysis. Usinganti-Mammastatin monoclonal antibodies, it has been demonstrated thatthere is a consistent difference in expression of this protein betweenmammary carcinoma cells and cells derived from normal mammaryepithelium. Mammastatin was recognized in normal human female mammarytissue as 44, 49, and 53 kD species by Western blot analysis withanti-Mammastatin monoclonal antibody 7G6. In mammary carcinoma cells,there was inconsistent recognition of a 44 kD species, but never 49 or53 kD immunoreactive forms. When the 49 and 53 kD forms are identifiedin normal cells they are phosphorylated. The 44 kD species is notphosphorylated. It is therefore possible to use immunoblot analysis todetermine if Mammastatin is phosphorylated by observing the expressionof the 44 and 49, and 53 kD species of Mammastatin.

EXAMPLE 15 Identification of Mammastatin in Human Sera

[0135] An enzyme-linked immunosorbant assay (ELISA) was established todetect Mammastatin, using the purified anti-Mammastatin monoclonalantibodies 6B8 and 3C6.

[0136] The antibody 6B8 was used to coat Immulon 1 96-well microtiterplates (Immulon Corp.) at a concentration of 10 μg/ml or 100 μl/well,for three (3) hours at room temperature, or overnight at 4° C. Plateswere blocked with 2% BSA (Sigma) in TBS (150 mM NaCl, 100 mM Tris pH7.4) for 30 minutes and were then incubated with either purifiedMammastatin or sample sera diluted 50% in 2% BSA solution for 1.5 hoursat 37° C. Microtiter plates were washed for 5 minutes, three times with300 μl/well of TBS plus 0.1% Triton X-100 before addition of secondantibody.

[0137] Second antibody was biotinylated 3C6. Antibody was biotinylatedby incubation with biotin, N-hydroxy succinimate ester (Sigma) in 0.1MNaHCO₃ for two hours at room temperature and 16 hours at 4° C. Antibodywas dialyzed into 1M NaCl, 50 mM Tris pH 7.4, 0.02% Azide (NaN₃, Sigma)before use or storage.

[0138] Biotinylated anti-Mammastatin antibody was added at 1 μl/ml and100 μl/well, in a 2% BSA/TBS solution and incubated for 1.5 hours at 37°C. Microtiter plates were washed 5 times for 5 minutes with TBS plus0.1% Triton X-100 as described above. Second antibody was identifiedwith alkaline phosphatase conjugated streptavidin (SouthernBiotechnology) and incubated for one hour at a dilution of 1/1000 in 2%BSA/TBS, 100 μl/well for all samples.

[0139] ELISA assays were developed colorometrically with PNPP(paranitrophenyl phosphate Sigma), 1 mg/ml in alkaline phosphatasebuffer (10 mM diethanolamine pH 9.5 (Sigma), 0.50 mM MgCl₂ (Sigma)).Microtitre plates were read on an ELISA reader at 405 nm at fifteenminute and thirty minute intervals.

[0140] Using chromatographically purified Mammastatin isolated from celllysates or conditioned media, a standard curve was established for theELISA indicating sensitivity of the assay for Mammastatin in the lownanogram range. (See FIG. 9). Quantitation of Mammastatin levels innormal human volunteer sera was performed in serum samples collected attwo day intervals for one month, from a volunteer. Mammastatin levels innormal human female sera were detectable by this assay and variedbetween about 10 and 50 ng/ml (FIG. 10).

[0141] Mammastatin levels were also measured in sera collected frombreast cancer patients. Patients diagnosed at the University of MichiganBreast Care Center with node negative breast cancer were tested forMammastatin expression in sera throughout the course of their treatment.The data are shown in FIG. 11 and summarized below. Serum samples werecollected from breast cancer patients during the entire course of theirtreatment on a hormonal cycling, combined modality protocol withCytoxin, Adriamycin, Methotrexate, and 5Fu. Serum was separated fromwhole blood, after clotting, by centrifugation and stored at −20° C.until use. ELISA assay using 150 μl serum at 50% in 0.5% NFDM induplicate were performed, using the 6B8 and 3C6 anti-Mammastatinantibodies in an enzyme linked—“sandwich” assay. The standard curve wasgenerated with chromatographically purified Mammastatin and wascomparable to that shown in FIG. 9.

[0142] Expression of Mammastatin varied among patients and fluctuatedduring the course of their treatment. It was consistently observed thatMammastatin levels became undetectable with progression to metastaticdisease.

[0143] Patients diagnosed with breast cancer had low levels ofMammastatin in serum at the time of diagnosis as compared with levels innormal patient serum. Mammastatin levels generally rose on the hormonalcycling, adjuvant chemotherapy protocol. Levels of Mammastatinfluctuated on this protocol. Mammastatin levels were undetectable inpatients with advanced disease, before death. The patient data sortedinto four groups, as shown in the table below.

[0144] I. Group of patients whose serum Mammastatin levels continued toraise during therapy.

[0145] II. Group of patients whose serum Mammastatin levels increasedinitially during therapy, but then became undetectable.

[0146] III. Group of patients whose serum Mammastatin levels rose duringtherapy, but then fluctuated widely.

[0147] IV. Group of patients who had low serum Mammastatin levels whichbecame undetectable with therapy. Summary of Mammastatin Levels inPatient Sera Group Number Days Followed Outcome I.  4 p 280 +/− 100Remission II. 14 p 500 +/− 220 Deceased III. 10 p 380 +/− 280 VariableIV.  5 p 290 +/− 150 Deceased

EXAMPLE 16 In vivo Efficacy of Mammastatin

[0148] CD-1 Nu/Nu homozygate, female, six week old mice (Charles River)were supplemented with Estrogen via slow release pellets, 0.72mg/pellet, 60 day release of 17-beta estradiol (Innovative Research#SE-121). Estrogen supplemented mice were injected with 3×10⁶ MCF-7cells 100 μl per injection in 60% matrigel. Two injections wereadministered, one per flank. After seven days of tumor cell growth,Mammastatin was administered. Test mice received 1, 2, or 5 μg ofMammastatin in production media at 2 day intervals for a period of sixweeks. Control mice were injected with BSA, or were not injected withtumor, but with the inhibitor alone.

[0149] Tumor size was measured at the point of greatest diameter atweekly intervals and averaged for treatment group. The results are shownin FIGS. 13A-13C, with tumor size plotted as the mean diameter±standarddeviation.

[0150] This animal study was repeated using MDA-231 tumor cells. Cellswere injected at a concentration of 2×10⁶ cells per injection asdescribed above for MCF-7 cells.

[0151] The results are shown in FIGS. 14A-14C.

[0152] The results shown were not as great as expected. The animals wereinjected by tail vein, resulting in less than the needed blood dose.Subsequent studies using intraperitoneal injection have resulted in moreeffective treatment. At doses of 5 ug/mouse and higher, tumor growth isabbrogated.

EXAMPLE 17 Retrovirus Expression of Mammastatin

[0153] The Mammastatin cDNA (2.4 kilobase (kb) insert) was subclonedinto a retroviral expression vector. The vector was used to transfect3T3 fibroblast cells. Transfected cells were harvested, lysed, and thecell lysate analyzed by Western Blot.

[0154] As shown in FIG. 12, 3T3 cells transfected with theMammastatin-carrying retrovirus, expressed phosphorylated Mammastatin.

[0155] The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. Since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended.

EXAMPLE 18 Expression of Mammastatin in Baclovirus and Cos 7 Cells

[0156]

[0157] Immunoblot Analysis: Cell lysates were probed with the 7G6,anti-mammastatin monoclonal antibody. A) 25 μg, NHMC-20, normal humanmammary cell lysate (control), B) 25 μg, Cos-7 cell lysates, transfectedwith pcDNA3 (control), C) 10 μg Cos-pMammB cell lysate, transfected withpcDNA3/mammastatin construct, D) 20 μg Cos-pMammB cell lysate. Repeated3 times with similar results.

[0158] Summary: Induction of recombinant mammastatin expression in Cos-7cells demonstrates that the mammastatin gene codes for authenticmammastatin. Furthermore, the observation that Cos-7 cells express thedifferent forms of mammastatin associated with phosphorylation of theprotein suggests that mammastatin will be phosphorylated and active whenproduced in eucaryotic cell lines other than human mammary cells. Stabletransfectants have been selected to allow perpetual synthesis ofrecombinant mammastatin.

EXAMPLE 19 Production of Mammastatin by Normal Human Breast EpithelialCells in Culture

[0159] Healthy breast tissue was obtained from reduction mammoplasty,sterile, and direct from the operating room. The tissue was minced understerile conditions in a laminar flow hood in a solution containing 4units per gram of type III collagenase (Life Sciences, Bethesda, Md.).The minced tissue was incubated overnight in a shaking water bath at 37°C. to allow collagenase digestion.

[0160] Collagenase digested breast tissue, a viscous fluid containing avariety of cell types and lipid released from adipose cells, wascentrifuged to separate lipid, aqueous solution, and other cell types.The collagenase-digested material was spun at 1000 rpm in a table topcentrifuge at room temperature for 5 minutes. Adipose cells and freelipid partitioned to the top half of the centrifuge tube, and werewithdrawn by aspiration and discarded. The aqueous supernatantpositioned above the cell pellet was also withdrawn by aspiration anddiscarded. The remaining cell pellet was washed with sterile solutionsof mammalian growth media, DMEM, pH 7.4. The washing was continued untilthe supernatant from the washes was no longer turbid (for example about4 washes). The washed cells were resuspended in growth media and allowedto settle by gravity for 30 minutes at 40° C. Because red blood cellsare enucleated and are less dense than nucleated epithelial cells, thisprocedure resulted in removal of the red blood cells from the sedimentedepithelial cells, by withdrawing the supernatant containing the redblood cells. This sedimentation procedure was repeated until no redcolor remained in the cell pellet, e.g., about 2 times. The remainingcell pellet was resuspended in a nutrient rich DMEM/F12 growth mediacontaining 5% equine serum, 10 μg/ml epidermal growth factor, 100 ng/mlof cholera toxin, 500 ng/ml hydrocortisone, 10 μg/ml insulin, 100units/ml penicillin and streptomycin, and 1 mM concentration of calciumchloride. Physiological concentrations of calcium helped to promote cellattachment and outgrowth in cell culture. The cell suspensions wereincubated in steril tissue culture flasks at 37° C. with a 5% CO₂concentration.

[0161] Initial cultures of normal breast tissue contain a mixed cellpopulation. The adipocytes, neurons, and vascular tissue aresignificantly reduced by the differential centrifugation processdescribed above. Connective tissue cells are present in signicantamounts. To remove non-epithelial cells, a differential attachmentmethod was used. Fibroblasts, neurons, and other cell types in breasttissue all attach to tissue culture plastic more rapidly than epithelialcells. In addition, all of these cell types are removed from tissueculture plastic by trypsin more rapidly than epithelial cells. To enrichthe cultures for breast epithelial cells, cultures beginning to form amonolayer (5-7 days after initial plating) are treated with atrypsin:EDTA solution (250:1) molar ratio. The majority of cells wereremoved within 5 minutes of incubation at 37° C. The remaining attachedcells were more than 90% epithelial breast cells. These cells wre savedand returned to the growth medium described above with 40 μM calciumchloride. The fibroblast cells were removed from the trypsinized cultureflasks, collected by centrifugation, resuspended in growth medium, andplated onto tissue culture plastic for 30 minutes at 37° C. The attachedcells were predominantly fibroblasts. The cells that did not attach weresignificantly enriched for fibroblast cells (50-80%). These suspendedcells were removed and allowed to settle in fresh tissue culture flasks.This process was repeated twice to obtain cell populations that werepredominantly epithelial. Because cholera toxin promotes epithelial cellgrowth and inhibits fibroblast growth, and because fibroblasts do notgrow well in reduced calcium, the cultures were approximately 100%epithelial within one week in the low calcium medium described above.These cultures of normal human mammary cells (NHMC) produced Mammastatininto the culture medium.

[0162] Nutrient medium used to grow the NHMC containes 5% equine serum,which is not acceptable for human injection. The equine serum proteinsmust either be purified away from the mammastatin protein, or the cellsgrown in medium devoid of the serum. Normal cells can only be maintainedin the absence of serum for about seven to ten days. In order to producea significant quantity of serum free mammastatin over a prolonged periodof time, the cells were alternately grown in serum-free andserum-containng medium.

[0163] NHMC were grown to complete confluence in growth media asdescribed above. The cells began to bud in solution as they grew, whencells covered the available surface of the flask. Budding cells werecollected and transferred to new flasks. Confluent flasks were rinsedthree times with sterile saline, with a five minute saline incubationbetween washes to remove serum protein. Cells were then provided withserum free “production medium” that was essentially the growth mediumdevoid of serum, cholera toxin, and hydrochortosone. Cells weremaintained on the production medium for about 4 days (96 hours), withcollection of the medium, and a return of the cells to growth medium forat least four days. The typical batch size for mammastain produced inthis way was 1-2 liters.

[0164] Mamastatin has also been produced in a Bioreactor, the Bioflow3000, New Brunswick Scientific. In this perfusion reactor, cells wereattached on tissue culture treated fibracell disks. The cell-attacheddisks were maintained in a basket in the reaction vessel and perfusedwith media. When NHMC were introduced to the reactor, they populate thefibracell disks and were fed by the perfusion of media. Conditionedmedia was harvested from the reactor and refrigerated.

EXAMPLE 20 Dot Blot Serum Assay for Mammastatin

[0165] Serum from a 25 year old healthy female was obtained and comparedwith serum from a breast cancer patient (Stage IV), an undiagnosedsibling of the patient, and from the patient's mother, whose family hasa history of breast cancer. The serum samples were compared in animmunoassay for the presence of Mammastatin. Blood samples from multiplebreast cancer patients taken on day of diagnosis were also analized inthe immunoassay. Normal human mammary cell (NHMC) conditioned media wasused as a standard control. Standard NHMC mammastatin containedapproximately 50 ng/ml as determined in a dot blot assay withmammastatin protein standard chromatographically purified.

[0166] Individual blood samples were collected into vacutainer tubes,and the serum separated from whole blood. Serum samples (250 or 500 μlvolume) were applied without dilution to nitrocellulose by suction usinga 96 well, S&S Dot-Blot manifold. Conditioned medium was prepared asdescribed for Example 19. Samples on the nitrocellulose filters werewashed with Triton X-100 in TBS, blocked with non-fat dry milk (5% inTBS) and incubated with 1 μg/ml of first anti-mammastatin antibody (7G6mouse IgM) in 5% non-fat dry milk for 1.5 hours at room temperature,followed by incubation with 1 μg/ml of second antibody (goat anti-mouseIgM conjugated to alkaline phosphatase) in 5% non-fat dry milk for onehour and room temperature. The alkaline phosphatase color reaction wasdeveloped using nitroblue-tetrazolium and BCIP.

[0167] As shown in FIG. 13, the amount of Mammastatin in the sample wasquantitated against the standard curve obtained from normal breast cellconditioned medium. Serum obtained from healthy females containedreadily detectable amounts of Mammastatin, as indicated by darklycolored blots, whereas serum from diagnosed breast cancer patients, andfrom undiagnosed family members showed little or no Mammastatin.

[0168] Additional samples were obtained from breast cancer patients onday of diagnosis, from healthy members of a breast cancer patient'sfamily, and from healthy females and males. The serum was processed asdescribed above in order to analize Mammastatin. The dot blots wereevaluated as “negative or low” or “positive or high” to indicate theintensity of the developed color reaction. Data are shown in thefollowing table. Sample Number Negative or Low Positive or High BreastCancer patient 89 83 (93%) 6 (7%)  Healthy female 11  2 (18%) 9 (82%)Healthy member of high  4  4 (100%) 0 risk family Male  3 2 1

EXAMPLE 21 Treatment of Human Breast Cancer Patients

[0169] Twenty-nine (29) Sage IV breast cancer patients with recurrentbreast cancer, who had failed, or were failing on chemotherapeuticregimes were given access to Mammastatin protein. The protein wasproduced as described above for Example 19, and provided in productionmedium, with the required dose in a 3 ml injection volume. Patientsadministered the protein intravenously according to their prescribedregimen. In general, one daily dose was injected. The selected dose wasthat which provided physiological amounts of Mammastatin in thepatient's bloodstream, e.g., 5-50 ng/ml in healthy women. The dosage andfrequency for each patient are indicated in the table below. PatientDose Number (ug) Schedule Result 1. 125 daily complete remissionfollowed by relapse* 2. 125 daily complete remission; pain if therapystopped 3. 75 daily non-responder* 4. 75 daily partial remission;possible immune reaction** 5. 75 daily non-responder* 6. 125 dailypartial remission 7. 125 daily partial remission* 8. 75 dailynon-responder* 9. 125 every third day complete remission 10. 125 dailynon-responder 11. 125 daily partial remission 12. 125 dailynon-responder# 13. 150 daily non-responder* 14. 75 daily non-responder#15. 125 daily partial remission 16. 125 daily partial remission 17. 75daily non-responder, infection** 18. 125 daily partial remission 19. 125daily partial remission 20. 125 daily partial remission 21. 125 everyother day non-responder**; alternative therapy 22. 125 every other daypartial remission 23. 125 every other day non-responder 24. 125 dailynon-responder 25. 125 daily non-responder 26. 125 daily partialremission 27. 125 daily partial remisssion 28. 125 every other daypartial remission 29. 125 daily % without % without % liver lung orliver Total Responders Responders involvement involvement 29 17 59 81 89

[0170] Of the group of 29 patients, six, having late stage liverdisease, did not survive. These six patients displayed clinical evidenceof liver failure before receiving Mammastatin, and were not helped bythe treatments. One patient showed signs of decreased jaundice beforeher liver failed, but all six of these patients appeared to die ofnitrogen toxicity common to patients with advanced liver cancer.

[0171] Of the remaining patients, two have died of their disease. Oneappeared to be disease-free after two months of Mammastatin therapy.This patient was removed from therapy, and relapsed within two months.The patient's disease was never brought back under control and she diedof liver involvement. The second patient died after 4 months oftreatment, having never shown any sign of response to the therapy. Therewas no sign of toxicity in any of these patients, although the dose ofMammastatin in these latter two patients was increased ten fold.

[0172] Of the 19 patients currently receiving Mammastatin therapy, themajority show signs of positive benefit and no signs of adversereaction. It is unclear if three of these patients are receiving anybenefit from Mammastatin. The other 16 patients show definite clinicalsigns of benefit including decreased tumor markers (CA15-3 and CA27-29)to normal levels, decreased size of palpable tumor masses, decreaseddisease as evidenced on MRI scan, and decreased pain. Several of thesepatients show improvement to the point of being considered disease free.However, it has consistently been observed that denying these patientsprotein for periods of three to five days results in resumption ofdisease activity as evidenced by increased pain, even in patients thatshow no signs of disease. Resumption of protein treatments decreases ofeliminates the symptoms of increased pain within 2-4 hours.

[0173] It has also been observed that Mammastatin levels in the blooddecline after long term treatment, suggesting a negative feedbacksystem. This decline in constant blood levels is successfully avoided byproviding Mammastatin daily for a period of about 28 days, followed by2-3 days without protein.

EXAMPLE 22 Recombinant Mammastatin for Human Therapy

[0174] Recombinant Mammastatin has been produced in Cos-7 monkey kidneycells, chinese hamster ovary (CHO) cells, and Sf9 insect cells bytransfecting the cells with a plasmid containing the Mammastatin cDNAsequence. The Mammastatin cDNA has been stably integrated into thegenomes of these producing cell lines, and secrete proteinimmunoreactive with growth inhibitory activity.

[0175] To produce Mammastatin from these cells and isolate the proteinfor human use, the cell lines are grown in serum free medium forapproximately 48 to 72 hours. The media is withdrawn and proteinpurified from conditioned medium, either by ion exchange chromatographyin Tris buffer, pH7.5, using a sodium chloride gradient from about 0.1 Mto about 0.5 M, collecting the Mammastatin fraction at about 0.2 M. Theprotein fraction is then dialized against normal saline, diluted ifnecessary, and filter sterilized.

[0176] In an alternative method, Mammastatin is produced as a fusionprotein in Cos7 or SF9 cells. The fusion protein contains a histidinetag (six histidine residues) and a Factor X proteinase cleavage site.The Mammastatin expressing cells are cultured, preferably in 1%serum-containing media, the conditioned media is collected and passedover a nickle chelating resin. The His-fusion protein adheres to thecolumn, is washed with 50 mM TRIS, pH 7.5, 0.1 M NaCl, and is slowlyeluted with TRIS-NaCl containing 10 Unit/ml Factor X proteinase. Thisliberates Mammastatin from the His fusion. Mammastatin is separated bymolecular sieve chromatography, or by ion exchange chromatography asdescribed above. TABLE 1 MAMMASTATIN DNA SEQUENCETGGGGCTCCACCCCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGGCACGAGCACGGTGAAGAGACATGAGAGGTGTAGAATCCGTGGGAGGCCCCCGGCGCCCCCCCGGTGTCCCCGCGAGGGGCCCGGGGCGGGGTCCGCCGGCCCTGCGGGCCGCCGGTGAAATACCACTACTCTTATCGTTTTTTCACTGACCCGGTCGAGCGGGGGGGCGAGCCCCGAGGGGCTCTCGCTTCTGGCGCCAAGCGCCCGGCCGCGCGCCGGCCGGGCGCGACCCGCTCCGGGGACAGTGCCAGGTGGGGAGTTTGACAGGGGCGGTACACCTGTCAAACGGTAACGCAGGTGTCCTAAGGCGAGCTCAGGGAGGACAGAAACCTCCCGTGGAGCAGAAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTGTAAGCGGGGCCTCACGATTCCTCTGACCTTTTGGGTTTTAAGCAGGAGGTGTCAGAAAAGTTACCACAGGGATAACTGGCTTGTGGCGGCCAAGCGTTCATTAGGACGTCGCTTTTTGATCCTTCGATGTCGGCTCTTCCTATCATTGTGTAGCAGAATTCACCAAGCGTTGGATTGTTCACCCACTAATAGGGAACGTGAGCTGGGTTTAGACCGTCGTGAGACAGGTTATTTTTACCCTACTGATGATGTGTTGTTGCCATGGTTATCCTGCTCAGTACGAGAGGAACCGCAGGTTCAGACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCATCTGTGGGATTATGACTGACGCTCTAAGTCATGAATCCCGCCCAGGCGGAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATTAGCCGGTCCCCCGCCTGTCCCCGCCGGCGGGCCGCCCCCCCCCCTCCACGCGCCCCGCGCGCGCGGGAGGGCGCGTGCCCCGCCGCGCGCCGGGACCGGGGTCCGGTGCGGAGTGCCCTTCGTCCTGGGAAACGGGGCGCGGCCGGAAAGGCGGCCGCCCCCTCGCCCGTCACGCACCGCACGTTCGTGCTCGTGCCGAATTCGGCACGAGTGCACCCATTCACAATATACATACAAGTGCATGTATCTTTATGATATAATGAATTCTTTTCCTTTGGGTAGATATCCAGTAGTGGGATTGCTAGATCACCTGGTAGTTCTATTTCTGGTTTATTTAGAAATCTTCATACTGATTTCCATAGAGGTTGTACAAATTTACATCCCTACCAAAGTGATTTTTTTAAATATGAAAGAATGGTCTGGAGAAATGCCCCTCATTAGTATCCCCCTTTTACCTCTCTACTGCAGAATGACTTCAAGGGGTACAGGTATTTACAAGTTTCATTATACAGACAAATTGAATATTGAAATTTTCTGCATAAGAGGCACAGATTTTAGGATTCAAAGTTGTATGAACAAGGACAAGTGCTCTAGGGACTTGCAAAGCTGGAATTGGAAATCTCAGATGAAATACATTTCTAGTAGTACCACCAGCATATATTCTACTGAATTGGCTTTTGTGATCATCATTAATACCTACTTATTAAAACTAATGAAAAGGGTTTATATCAAATATACTTTAAGGTATAAAAATCAAATTATAGGTAAAGCTGTTTTCTTTAGCATTTTAATTTCAAAACATAAAATAGCTACCGTCTATTGGGCATTTATACTGTACCAGACACTGTGTTTGTCACATTTCAAAAATGTTCTCATGGTAATGTTCACAATAATTCTGTCGGGTGAGAAAATAGTCTTACCGTAGTAAGACTATTCAGTAAAACGAAACCTCTGAACCTTGGAGTTCAACTTGCGCAAAGTTAGTAACAGGACTAGGACTTGAACCTGAACCATCACACTCCAGATCTCTCCATACCACACTGCTAGCACATGTGCCTGTCATCTTATTCCTGGCTCCCTTTTTTATTTCCTTTCCCTTCCTCCCACAACCCCTTTTTCCCCCCATTTCTTTCTTTCTTTTTATTTGTTAATTACATAACTAATACATGTTTATCAGAACAATTGATATAGCACAAAAGGATATAAAGTACGGGGGAGTGATAGCTCATCCCTGTAATCCTAGCACTTTGGAAGGCCAAGGCAGGCAGATCACTTTGAGTCCAGAGTTCGAGACCAGCCTGGGCAACATGGTG:AAACCCTGTCTCTACAAAAAAATACAAAAAATTTAGCCGGGCGTGCTGGCACACACCTGTAGTCTCAGCTACTCTGAGGGCTGAGGTGGGAAGATTGATTGAGCCCAGGAGGTGGAAGCTGCAGCAGTGCGCTGAGATTGCGCCATTGCACTCCAGCCTGGGTGAGAGAGAGACCCTGTCTCCAAAAAAAAAAAAAAAAAAAAAAA

We claim:
 1. A substantially purified and isolated nucleic acidcomposition comprising the coding sequence of nucleic acid sequence ofSeq. ID NO:
 1. 2. A substantially purified and isolated proteincomprising the amino acid sequence of Seq. ID NO:
 2. 3. A recombinantprotein produced from the nucleic acid sequence of claim
 1. 4. A plasmidor vector comprising the coding sequence of nucleic acid sequence ofclaim
 1. 5. A diagnostic kit for the determination of Mammastatincomprising the composition of claim
 1. 6. A diagnostic kit for thedetermination of Mammastatin comprising anti-Mammastatin antibodies andMammastatin standards.
 7. A pharmaceutical composition comprising thecomposition of claim
 2. 8. A composition comprising the human nucleicacid sequence insert of pMammB, ATCC No.
 97451. 9. A method for thediagnosis or monitoring of mammary cell carcinoma comprising: analyzinga patient's blood or tissue for the presence of Mammastatin protein; andcorrelating the absence or reduction of Mammastatin as compared with anormal control with mammary cell carcinoma.
 10. The method of claim 8,wherein said analyzing is determining the presence or absence of 53 kDa,49 kDa, and 44 kDa Mammastatin in the patient sample, and wherein saidcorrelating is correlating the absence or reduction of 53 kDa or 49 kDaMammastatin as compared with a normal control with the presence ofmammary cell carcinoma.
 11. A method of monitoring the level offunctional Mammastatin in a human subject comprising the steps of:analyzing a biological sample of body fluid or tissue of the subject forthe presence of three molecular weight forms of Mammastatin having theapproximate sizes 53 kDa, 49 kDa and 44 kDa; and correlating a reducedamount of 53 kDa or 49 kDa or 44 kDa Mammastatin relative to a normal orprior patient sample control with a reduced amount of functionalMammastatin.
 12. A method of treating a patient suffering from breastcancer comprising the steps of: administering to the patient a mammarycell growth-inhibiting amount of Mammastatin.
 13. The method of claim11, wherein said administering step is repeated to maintaintherapeutically effective levels of Mammastatin in vivo.
 14. A methodfor inhibiting the growth of human mammary cells comprisingadministering to said cells a growth-inhibiting amount of Mammastatin.15. The method of claim 11, wherein said Mammastatin is encoded by thecomposition of claim
 1. 16. The method of claim 11, wherein saidMammastatin comprises the composition of claim
 2. 17. The method ofclaim 11, wherein said Mammastatin comprises the composition of claim 7.